Antithrombotic agents

ABSTRACT

This invention relates to L-Arginine aldehyde derivatives, pharmaceutical formulations containing those compound and methods of their use as thrombin inhibitors.

BACKGROUND OF THE INVENTION

This invention relates to thrombin inhibitors which are usefulanticoagulants in mammals. In particular it relates to L-Argininealdehyde derivatives having high antithrombotic activity, anticoagulantactivity, and oral bioavailability.

The process of blood coagulation, thrombosis, is triggered by a complexproteolytic cascade leading to the formation of thrombin. Thrombinproteolytically removes activation peptides from the Aα-chains andBβ-chains of fibrinogen, which is soluble in blood plasma, initiatinginsoluble fibrin formation.

Anticoagulation is currently achieved by the administration of heparinsand coumarins.

Parenteral pharmacological control of coagulation and thrombosis isbased on inhibition of thrombin through the use of heparins. Heparinsact indirectly on thrombin by accelerating the inhibitory effect ofendogenous antithrombin III (the main physiological inhibitor ofthrombin). Because antithrombin III levels vary in plasma and becausesurface-bound thrombin seems resistant to this indirect mechanism,heparins can be an ineffective treatment. Because coagulation assays arebelieved to be associated with efficacy and with safety, heparin levelsmust be monitored with coagulation assays (particularly the activatedpartial thromboplastin time (APTT) assay). Coumarins impede thegeneration of thrombin by blocking the posttranslationalgamma-carboxylation in the synthesis of prothrombin and other proteinsof this type. Because of their mechanism of action, the effect ofcoumarins can only develop slowly, 6-24 hours after administration.Further, they are not selective anticoagulants. Coumarins also requiremonitoring with coagulation assays (particularly the prothrombin time(PT) assay).

Recently, interest in small synthetic peptides that are recognized byproteolytic enzymes in a manner similar to that of natural substrateshas grown. Tripeptide aldehydes such as D-Phe-Pro-Arg-H,Boc-D-Phe-Pro-Arg-H, and D-MePhe-Pro-Arg-H, Bajusz et al., J. Med.Chem., 33, 1729-1735 (1990) demonstrate potent direct inhibition ofthrombin. Many investigators have synthesized analogs in an effort todevelop pharmaceutical agents, for example Shuman et al., J. Med. Chem.,36, 314-319 (1993).

Although the heparins and coumarins are effective anticoagulants, and nodrug has yet emerged from the known tripeptide aldehydes, and despitethe continuing promise for this class of compounds, there exists a needfor anticoagulants that act selectively on thrombin, and independent ofantithrombin III, exert inhibitory action shortly after administration,preferably by an oral route, and do not interfere with lysis of bloodclots, as required to maintain hemostasis.

The present invention is directed to the discovery that the compounds ofthe present invention, as defined below, are potent thrombin inhibitorsthat may have high bioavailability following oral administration.

Accordingly, it is a primary object of the present invention to providenovel L-Arginine aldehyde derivatives that are potent thrombininhibitors useful as anticoagulants.

Other objects, features, and advantages will be apparent to thoseskilled in the art from the following description and claims.

SUMMARY OF THE INVENTION

The present invention provides thrombin inhibiting compounds having theFormula ##STR1## wherein

X is ##STR2##

Q is --OH, C₁ -C₄ alkoxy, or --NH--A;

A is hydrogen, C₁ -C₄ alkyl, acetyl, CF₃ C(O)--, CF₃ CF₂ C(O)--, R"SO₂--, benzyloxycarbonyl, or t-buryloxycarbonyl;

R' is hydrogen, C₁ -C₄ alkyl, phenyl, or benzyl;

R" is C₁ -C₄ alkyl, --(CH₂)_(d) --COOH, or unsubstituted or substitutedaryl or heteroaryl (Ar), where aryl is phenyl or naphthyl, andheteroaryl is a 5- or 6-membered unsubstituted or substituted aromaticheterocyclic ring, having one or two heteroatoms which are the same ordifferent and which are selected from sulfur, oxygen and nitrogen, or a9- or 10-membered unsubstituted or substituted fused bicyclic aromaticheterocyclic group having one or two heteroatoms which are the same ordifferent and which are selected from sulfur, oxygen and nitrogen;

d is 1, 2, or 3;

m is 0, 1, or 2;

n is 0, 1, or 2;

Y is ##STR3##

R is methyl or ethyl; and

Z is hydrogen, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, hydroxy, halo, or R_(a) SO₂NH--, where R_(a) is C₁ -C₄ alkyl;

and pharmaceutically acceptable salts and solvates thereof.

In addition to the compounds of Formula I, the present inventionprovides pharmaceutical formulations comprising a compound of Formula Iin association with a pharmaceutically acceptable carrier, diluent orexcipient.

The present invention also provides a method of inhibiting thrombosis inmammals comprising administering to a mammal in need of treatment, anantithrombotic dose of a compound of Formula I.

The present invention further provides a method of inhibiting thrombincomprising administering to a mammal in need of treatment, a thrombininhibiting dose of a compound of Formula I.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to new inhibitors of thrombin, pharmaceuticalcompositions containing the compounds as active ingredients, and the useof the compounds as anticoagulants for prophylaxis and treatment ofthromboembolic diseases such as venous thrombosis, pulmonary embolism,arterial thrombosis, in particular myocardial ischemia, myocardialinfarction and cerebral thrombosis, general hypercoagulable states andlocal hypercoagulable states, such as following angioplasty and coronarybypass operations, and generalized tissue injury as it relates to theinflammatory process.

The term "alkyl" by itself or as part of another substituent means astraight or branched chain alkyl radical having the stated number ofcarbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl and sec-butyl.

The term "alkoxy" means a straight or branched chain alkyl radicalhaving the stated number of carbon atoms bonded to the parent moiety byan oxygen atom. The term "halo" means chloro, fluoro, bromo or iodo. Theterm "acetyl" means CH₃ --C(O)--. The term "t-butyloxycarbonyl" means(CH₃)₃ C--O--C(O)-- and is abbreviated "Boc". The term"benzyloxycarbonyl" means C₆ H₅ CH₂ --O--C(O)-- and is abbreviated"Cbz".

The term "5- or 6-membered heterocyclic ring" means any 5- or 6-memberedring that will afford a stable structure containing one or two nitrogenatoms; one sulfur atom; one oxygen atom; one nitrogen and one sulfuratom; or one nitrogen and one oxygen atom. The 5-membered ring has oneor two double bonds and the 6-membered ring has two or three doublebonds. Such heterocyclic systems include furyl, thienyl, pyrrolyl,pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyranyl,pyridinyl, pyrimidinyl, pyrazinyl, oxazinyl and thiazinyl.

The term "9- or 10-membered heterocyclic ring" means any bicyclic groupin which any of the above 5- or 6-membered rings is fused to a benzenering or another 6-membered heterocyclic ring as defined above that willafford a stable structure. These heterocyclic systems include indolyl,benzothienyl, benzofuryl, benzoxazolyl, benzoisoxazolyl, benzopyrazolyl,quinolinyl, isoquinolinyl, benzimidazolyl and benzothiazolyl.

It will be appreciated that many of the above heterocycles may exist intautomeric forms. All such forms are included within the scope of thisinvention.

All of the aryl or heteroaryl groups listed for the definition of Ar areunsubstituted or substituted with one or two substituents that willafford a stable structure independently selected from halo, hydroxyl, C₁-C₄ alkyl, C₁ -C₄ alkoxy, amino (--NH₂), substituted amino (--NHR¹),--(CH₂)_(k) COOH, mercapto, and substituted thio (--S(O)_(h) R¹), and R¹is C₁ -C₄ alkyl, C₁ -C₄ alkoxy, (C₁ -C₄ alkyl)S(O)_(h) --, amino, C₁ -C₄alkylamino, (C₁ -C₄ alkyl)--C(O)--, or (C₁ -C₄ alkyl)SO₂ NH--, h is 0, 1or 2, and k is 0, 1, 2, 3, or 4.

In the representation of Formula I, the carbonyl functionality of groupX is attached to the amine functionality of the Y group. The carbonylfunctionality of Y is then attached to the amino group drawn in FormulaI.

The group ##STR4## where Z and A are both hydrogen, is referred to attimes herein as phenylglycyl and abbreviated Phg. Compounds wherein Ais, e.g., methyl, are referred to as the N.sup.α ethyl-phenylglycylgroup and abbreviated MePhg. Substituted compounds wherein Z is otherthan hydrogen are referred to by the type and position of thesubstituent group, e.g., 3'-chlorophenylglycyl or Phg (3-Cl).

The group ##STR5## where Z and A are both hydrogen, is referred to attimes herein as phenylalanyl and abbreviated Phe. Compounds wherein Ais, e.g., methyl, are referred to as the N.sup.α methyl-phenylalanylgroup and abbreviated MePhe. Substituted compounds wherein Z is otherthan hydrogen are referred to by the type and position of thesubstituent group, e.g., 3'-chlorophenylalanyl or Phe (3-Cl).

The groups ##STR6## when R' is hydrogen, are referred to at times hereinas 1- and 3-tetrahydro-isoquinolinecarboxylate, respectively, and arerespectively abbreviated 1-Tiq and 3-Tiq.

The groups ##STR7## when R' is hydrogen, are referred to at times hereina 1- and 3-perhydro-isoquinolinecarboxylate, respectively, and arerespectively abbreviated 1-Piq and 3-Piq. As indicated by the crookedlines, various ring fusion isomers of these substituents exist--thisinvention contemplates any individual isomer and combinations thereof.

The groups ##STR8## are referred to as prolinyl andazetidine-2-carboxyl, respectively, and are respectively abbreviated Proand Azt.

The asterisks in Formula I and substituent Y denote a chiral center thatis (L). The asterisk in substituent X denotes a chiral center that is(D) or (DL).

In addition, diastereomers may exist depending upon branching of alkylsubstituents. The compounds of the present invention include mixtures oftwo or more diastereomers as well as each individual isomer.

The following compounds illustrate compounds contemplated within thescope of Formula I:

D-phenylalanyl-L-prolinyl-L-(α-methyl)arginine aldehyde;

D-phenylglycyl-L-azetidine-2-carbonyl-L-(α-methyl)arginine aldehyde;

D-1,2,3,4-tetrahydroisoquinolinyl-3-carbonyl-L-azetidine-2-carbonyl-L-(α-ethyl)arginine aldehyde;

D-2-naphthylalanine-L-azetidine-2-carbonyl-L-(α-methyl)argininealdehyde; and

D-perhydroisoquinolinyl-l-carbonyl-L-prolinyl-L-(α-methyl)argininealdehyde.

Preferred compounds of the present invention are those compounds ofFormula I where A is hydrogen, X is MePhe, 1- or 3-Tiq, or 1- or 3-Piq,and Y is as defined above for Formula I, and pharmaceutically acceptablesalts and solvates thereof.

As mentioned above, the invention includes pharmaceutically acceptablesalts of the compounds defined by the above Formula I. A particularcompound of this invention can possess one or more sufficiently basicfunctional groups, and accordingly react with any of a number ofinorganic and organic acids, to form a pharmaceutically acceptable salt.Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suchpharmaceutically acceptable salts thus are the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like. Preferredpharmaceutically acceptable acid addition salts are those formed withmineral acids such as hydrochloric acid, hydrobromic acid and sulfuricacid.

The compounds of the present invention are known to form hydrates andsolvates with appropriate solvents. Preferred solvents for thepreparation of solvate forms include water, alcohols, tetrahydrofuran,DMF, and DMSO. Preferred alcohols are methanol and ethanol. Otherappropriate solvents may be selected based on the size of the solventmolecule. Small solvent molecules are preferred to facilitate thecorresponding solvate formation. The solvate or hydrate is typicallyformed in the course of recrystallization or in the course of saltformation. One useful reference concerning solvates is Sykes, Peter, AGuidebook to Mechanism in Organic Chemistry, 6th Ed (1986, John Wiley &Sons, New York). As used herein, the term "solvate" includes hydrateforms, such as monohydrates and dihydrates.

The compounds of Formula I are prepared by known methods of peptidecoupling. According to one such method the acid P-X'-COOH, where--X'-C(O)-- has the same meaning as --X -- defined for Formula I, and Pis an amino protecting group, is coupled with a carboxy protectedproline (or azetidine-2-carboxylic acid) to form the dipeptide (a). Thecarboxy protecting ester group of the proline or azetidine moiety isthen removed (deblocked or de-esterified) and the free acid form of thedipeptide (b) is coupled with the lactam form of (αR)arginine (d). Theabove reaction sequence is illustrated by the following Scheme 1:##STR9## wherein P represents an amino protecting group and n is 1 or 2.

The coupled (αR)Arg(P) lactam product (c) is reacted with a hydridereducing agent, preferably lithium aluminum hydride or lithiumtri-tert-butoxyaluminohydride, in an inert solvent or mixture ofsolvents to reduce the lactam ring and provide the tripeptide in thearginine aldehyde form represented by the formula ##STR10## wherein (P)represents amino protecting groups.

The protecting groups are removed by procedures known to those skilledin the art such as hydrogenation over a metal catalyst.

The lactam form of (αR)arginine is obtained by intramolecular couplingof amino protected αR-substituted arginine [(αR)Arg-OH]. For example,Boc-(αR)Arg(Cbz)OH represented by the formula ##STR11## where Boc ist-butyloxycarbonyl and Cbz is benzyloxycarbonyl, is first converted toan active ester form, such as an active mixed anhydride, with achloroformate ester, e.g. ethyl chloroformate to isobutyl chloroformate.The ester formation is carried out in the presence of a tertiary aminesuch as N-methylmorpholine. Addition of further or another tertiaryamine base, such as triethylamine or diisopropylethylamine, effects theinternal acylation to provide the lactam form of the diamino protectedarginine as shown below ##STR12## Prior to use in the coupling with theP-X'-(C=O)-Pro(or Azt)-OH as shown in the above scheme, the Boc or otheramine protecting group is selectively removed with trifluoroacetic acidor HCl to provide the requisite free amino group.

The coupling of a P-X'-COOH compound with a proline or azetidinecarboxylic ester is carried out by first protecting the amino group ofthe amino acid. Conventional amino protecting groups commonly used fortemporary protection or blocking of the amino group are employed.

The amino-protecting group refers to substituents of the amino groupcommonly employed to block or protect the amino functionality whilereacting other functional groups on the compound. Examples of suchamino-protecting groups include the formyl group, the trityl group, thephthalimido group, the trichloroacetyl group, the chloroacetyl,bromoacetyl and iodoacetyl groups, urethane-type blocking groups such asbenzyloxycarbonyl, t-butoxycarbonyl, 4-phenylbenzyloxycarbonyl,2-methylbenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,4-fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl,3-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl,4-cyanobenzyloxycarbonyl, 2-(4 -xenyl) isopropoxycarbonyl,1,1-diphenyleth -1-yloxycarbonyl, 1,1-diphenylprop-1-yloxycarbonyl,2-phenylprop- 2-yloxycarbonyl, 2-(p-toluyl) prop-2-yloxycarbonyl,cyclopentanyloxycarbonyl, 1-methylcyclopentanyloxycarbonyl,cyclohexanyloxycarbonyl, 1-methylcyclohexanyloxycarbonyl,2-methylcyclohexanyloxycarbonyl, 2-(4-toluylsulfonyl)ethoxycarbonyl,2-(methylsulfonyl)ethoxycarbonyl, 2-(triphenylphosphino)ethoxycarbonyl,9-fluoroenylmethoxycarbonyl ("FMOC"), 2-(trimethylsilyl) ethoxycarbonyl,allyloxycarbonyl, 1-(trimethylsilylmethyl) prop-1-enyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropylmethoxycarbonyl, 4-(decyloxy)benzyloxycarbonyl,isobornyloxycarbonyl, 1-piperidyloxycarbonyl and the like; thebenzoylmethylsulfonyl group, the 2-(nitro)phenylsulfenyl group, thediphenylphosphine oxide group, and the like amino-protecting groups. Thespecies of amino-protecting group employed is not critical so long asthe derivatized amino group is stable to the condition of subsequentreaction(s) on other positions of the molecule and can be removed at theappropriate point without disrupting the remainder of the molecule.Preferred amino-protecting groups are the benzyloxycarbonyl,allyloxycarbonyl, t-butoxycarbonyl, and trityl groups. Similaramino-protecting groups used in the cephalosporin, penicillin andpeptide art are also embraced by the above terms. Further examples ofgroups referred to by the above terms are described by J. W. Barton,"Protective Groups in Organic Chemistry", J. G. W. McOmie, Ed., PlenumPress, New York, N.Y., 1973, Chapter 2, and T. W. Greene, "ProtectiveGroups in Organic Synthesis", John Wiley and Sons, New York, N.Y., 1981,Chapter 7. The related term "protected amino" defines an amino groupsubstituted with an amino-protecting group discussed above.

In carrying out the coupling reaction an ester protecting group forproline is employed which is removable by conditions under which theamino protecting group remains intact. The amino protecting group of theacylating acid P-X'-COOH thus remains in place for protection of theamino group during the subsequent coupling with the arginine lactamcompound to form (c).

The carboxy protecting ester group as used in the specification refersto one of the ester derivatives of the carboxylic acid group commonlyemployed to block or protect the carboxylic acid group while reactionsare carried out on other functional groups on the compound. Examples ofsuch carboxylic acid protecting groups include C₁ -C₃ alkyl, benzyl,4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl,2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl,pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl,4,4'-dimethoxybenzhydryl, 2,2', 4,4'-tetramethoxybenzhydryl, t-butyl,t-amyl, trityl, 4-methoxytrityl, 4,4'-dimethoxytrityt, 4,4',4"-trimethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl,t-butyldimethylsilyl, phenacyl, 2,2,2 -trichloroethyl,β-(trimethylsilyl)ethyl, β-(di(n-butyl)methylsilyl) ethyl,p-toluenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)-prop-1-en-3-yl, and like moieties. The speciesof carboxy-protecting group employed is not critical so long as thederivatized carboxylic acid is stable to the conditions of subsequentreaction(s) on other positions of the molecule and can be removed at theappropriate point without disrupting the remainder of the molecule. Inparticular, it is important not to subject the carboxy-protectedmolecule to strong nucleophilic bases or reductive conditions employinghighly activated metal catalysts such as Raney nickel. (Such harshremoval conditions are also to be avoided when removing amino-protectinggroups discussed below.) Further examples of these groups are found inE. Haslam, "Protective Groups in Organic Chemistry", J. G. W. McOmie,Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T. W. Greene,"Protective Groups in Organic Synthesis", John Wiley and Sons, New York,N.Y., 1981, Chapter 5.

The compounds of Formula I can also be prepared by first synthesizing aPro- (or Azt)-(αR)Arg dipeptide precursor and then reacting with aprotected X-substituent. According to one such method, the cyclic lactamform of (αR)arginine (d) is prepared and coupled with an amino protectedproline or azetidine-2-carboxylic acid (g) as shown below to afford thedipeptide (h) ##STR13## where P represents an amino protecting groupsuch as the benzyloxycarbonyl (Cbz) group, t-butoxycarbonyl (Boc),p-toluenesulfonyl, and the like. Preferably the amino protecting groupused is removable by hydrogenation or treatment with mild acid (e.g.trifluoroacetic acid) or a strong acid (e.g. HCl). Examples of othersuitable amino protecting groups are provided in "Protective Groups inOrganic Synthesis", Second Edition, by T. W. Greene and Peter G. M.Wuts, Chapter 7, page 309-405 (1991), John Wiley & Sons, Inc.,publishers, incorporated herein by reference in its entirety. The Boc,or other suitable protecting group, is removed from the azetidine ringnitrogen which is then acylated with the desired amino acid acyl groupto afford the tripeptide shown below. ##STR14## The coupled (αR)Arg(P)lactam product (c) is reduced and the protecting groups are removed asdescribed earlier.

The coupling of an P-X'-COOH compound is carried out by first protectingthe amino group of the amino acid. Conventional amino protecting groupscommonly used for temporary protection or blocking of the amino groupare employed. Examples of such protecting groups are described above.

The coupling reactions described above are carried out in the coldpreferably at a temperature between about -20° C. and about 15° C. Thecoupling reactions are carried out in an inert organic solvent such asdimethylformamide, dimethylacetamide, tetrahydrofuran, methylenechloride, chloroform, and like common solvents or a mixture of suchsolvents. Generally anhydrous conditions are used when, in the couplingreaction, an active ester of the acylating acid is used.

The lactam intermediates (d) and (f) are prepared via standardtechniques of organic chemistry as summarized in the following scheme:##STR15##

According to the above sequence, benzophenone imine can be used toprotect alanine (R=methyl) or 2-aminobutyric acid (R=ethyl) to providethe protected amino acid (h). The precursor to the arginine group isintroduced by alkylating (h) with an α-halopropionitrile to give (i).After deblocking the amine and reblocking with a protecting group moresuitable for the subsequent reactions, such as a Boc group (e.g.,intermediate (j)),the amino acid is first deesterified (k) and reducedto give the ornithine intermediate (1). Treatment with O-methylisoureain base provides the arginine derivative (m). The primary amine isblocked, e.g., with a Cbz functionality, to give (e) which is thencyclized as described above to give (f). Removing the Boc protectinggroup yields a primary amine (d') which is then used to couple to themono- or di-peptide. As will be appreciated by skilled artisans, one maychoose other protecting groups so long as they serve the purpose ofprotecting the functional group during subsequent chemistry but can alsobe removed under appropriate conditions and in an appropriate order toallow for subsequent transformations.

The compounds of the invention are isolated best in the form of acidaddition salts. Salts of the compounds of Formula I formed with acidssuch as those mentioned above are useful as pharmaceutically acceptablesalts for administration of the antithrombotic agents and forpreparation of formulations of these agents. Other acid addition saltsmay be prepared and used in the isolation and purification of thepeptides. For example, the salts formed with the sulfonic acids such asmethanesulfonic acid, n-butanesulfonic acid, p-toluenesulfonic acid andnaphthalenesulfonic acid may be so used.

The preferred method for purifying the compounds of Formula I, while atthe same time preparing a desired stable salt form, is that described inU.S. Pat. No. 5,250,660. According to the method, stable sulfates orhydrochlorides are provided by preparative purification over C₁₈reversed-phase chromatography in which the aqueous component comprisessulfuric acid or hydrochloric acid at pH 2.5 and acetonitrile as theorganic component. The pH of the acidic eluant is adjusted to betweenabout pH 4 and about 6 with an anion exchange resin in the hydroxyl forme.g. Bio-Rad AG-1×8. After adjustment of the pH, the solution oftripeptide sulfate or hydrochloride salt is lyophilized to provide thepure salt in dry powder form. In an example of the process, crudeD-MePhe-Pro-Arg(αMe)-H sulfate is dissolved in water and the solution isloaded on Vydac C₁₈ RP-HPLC 5 cm×50 cm column. A gradient of 2-10% B(A=0.01% H₂ SO₄ ; B=acetonitrile) over 10 hours is used. Multiplefractions are collected and those containing product as determined byanalytical RP-HPLC are pooled. The pH of the pooled fractions isadjusted to pH 4.0-4.5 with AG-1×8 resin in hydroxide form (Bio-Rad,3300 Ragatta Blvd., Richmond, Calif. 94804). The solution is filteredand the filtrate is lyophilized to provide the pure D-,L-,L-, tripeptidein the form of the sulfate salt.

The optically active isomers of the diastereomers of the X substituentare also considered part of this invention. Such optically activeisomers may be prepared from their respective optically activeprecursors by the procedures described above, or by resolving theracemic mixtures. This resolution can be carried out by derivatizationwith a chiral reagent followed by chromatography or by repeatedcrystallization. Removal of the chiral auxiliary by standard methodsaffords substantially optically pure isomers of the compounds of thepresent invention or their precursors. Further details regardingresolutions can be obtained in Jacques, et al., Enantiomers, Racematesand Resolutions, John Wiley & Sons, 1981.

The compounds employed as initial starting materials in the synthesis ofthe compounds of this invention are well known and, to the extent notcommercially available, are readily synthesized by standard procedurescommonly employed by those of ordinary skill in the art.

The following Examples are provided to further describe the inventionand are not to be construed as limitations thereof.

The R_(f) values in the following examples unless otherwise stated, weredetermined by silica gel thin layer chromatography using Kieselgel60F-254 (Merck, Darmstadt) in the following solvent systems:

(A) Chloroform-Methanol-Acetic Acid 135:15:1

(B) Ethyl acetate-Acetic Acid-Absolute Alcohol 90:10:10

(C) Chloroform-Methanol-Acetic Acid 90:30:5

The abbreviations used in the examples have the following meanings.

Amino acids: Arg=arginine, Pro=proline, Phe=phenylalanyl,1-Tiq=1-tetrahydroisoquinolinecarboxylate

Boc=t-butyloxycarbonyl

Cbz=benzyloxycarbonyl

DMF=dimethylformamide

EtOAc=ethyl acetate

Et₂ O=diethyl ether

FAB-MS=fast atom bombardment mass spectrum

THF=tetrahydrofuran

TLC=thin layer chromatography

Unless otherwise stated, pH adjustments and work up are with aqueousacid or base solutions.

EXAMPLE 1 D-N-methylphenylalanyl-L-prolinyl-L-α-methylarginine aldehyde(D-MePhe-Pro-Arg (αMe)-H) ##STR16## A) Preparation of ethyl N.sup.αdiphenylmethylene-L-alaninate.

Benzophenone imine (53.4 g, 286 mmol) was dissolved in methylenechloride (400 mL) and stirred at room temperature. To the solution wasadded L-alanine ethyl ester (43.9 g, 286 mmol) and the reaction stirredat room temperature for 48 hours. The reaction was washed 3 times withwater (200 mL). The organic layer was separated, dried (MgSO₄), and thefiltrate was concentrated in vacuo to give a clear oil. The oil wascrystallized from pentane to give the title compound (72.1 g, 90%),FAB-MS 282 (MH⁺).

Analysis for C₁₈ H₁₉ NO₂ :

Calculated: C, 76.84; H, 6.81; N, 4.98.

Found: C, 76.73; H, 6.68; N, 5.22.

B) Preparation of ethyl N.sup.αdiphenylmethylene-DL-(α-propionitrile)alaninate.

A solution of ethyl N.sup.α diphenylmethylene-L-alaninate (20 g, 71.2mmol) in anhydrous THF (300 mL) was added to 18-crown-6 (18.8 g, 71.2mmol), potassium hydride (17.8 g, 106.8 mmol), and THF (100 mL), andstirred under an inert atmosphere. The reaction was cooled to 0° C. andbromopropionitrile (8.9 mL, 106.8 mmol) dissolved in THF (20 mL) wasadded dropwise. The reaction was warmed to room temperature and stirred(2 hours). To the reaction was added a solution containing glacialacetic acid (6.5 mL), water (25 mL), and THF (20 mL) dropwise. Thereaction was diluted with ethyl acetate and water. The organic layer wasseparated, washed 3 times with water, and dried (MgSO₄). The filtratewas concentrated in vacuo to give an oil. The crude oil was dissolved inmethylene chloride/cyclohexane and chromatographed over silica gel. Astep gradient system consisting of (A) hexane and (B) EtOAc was used toelute the pure compound. The gradient used was an increasingconcentration of EtOAc from 0% to 25%. Fractions were collected andpooled on the basis of TLC profile. The combined fractions wereconcentrated in vacuo to give a clear oil of the title compound (15.5 g,65%), FAB-MS 335 (MH⁺).

C) Preparation of ethyl DL-(α-propionitrile)alaninate.

A solution of ethyl N.sup.αdiphenylmethylene-DL-(αpropionitrile)-alaninate (15.2 g, 45.4 mmol) indiethyl ether (90 mL) was cooled to 0° C. To the reaction was added 1 NHCl (54 mL) dropwise. The reaction was warmed to room temperature andstirred (24 hours). The aqueous layer was separated and extracted 3times with diethyl ether. The aqueous layer separated and concentratedin vacuo to give a clear oil of the title compound (9.7 g, 100%), FAB-MS171 (MH⁺).

D) Preparation of ethyl N.sup.αt-butyloxycarbonyl-DL-(α-propionitrile)alaninate.

A solution of ethyl DL-(α-propionitrile)alaninate (7.8 g, 37.8 mmol) inTHF (50 mL) was charged with diisopropylethylamine (6.6 mL, 37.8 mmol)and stirred at room temperature. Di-tert-butyl dicarbonate (9.6 mL, 41.6mmol) was added and the reaction stirred (24 hours). The reaction wasdiluted with EtOAc/water and the organic layer separated. The organicsolution was washed 2 times with 0.1 N HCl, dried (MgSO₄), and thefiltrate concentrated in vacuo to give an oil. The oil was crystallizedfrom pentane to give the title compound (7.45 g, 73%), FAB-MS 271 (MH⁺).

Analysis for C₁₃ H₂₂ N₂ O₄ :

Calculated: C, 57.76; H, 8.00; N, 10.36;

Found: C, 57.76; H, 8.30; N, 10.45.

E) Preparation of N.sup.αt-butyloxycarbonyl-DL-(α-propionitrile)alanine.

A solution of ethyl N.sup.αt-butyloxycarbonyl-DL-(α-propionitrile)-alaninate (12.6 g, 46.4 mmol) inTHF (100 mL) and water (58 mL) was stirred and cooled (0° C.). To thereaction was added 1 N NaOH (47 mL, 47 mmol) and the solution wasstirred at 0° C. for 30 minutes and at room temperature (4 hours). Theorganic solvent was evaporated in vacuo, and EtOAc (100 mL) and water(100 mL) were added to the residue. The aqueous layer was separated, thepH of the solution was adjusted to 2.8 with 3 N HCl, and ethyl acetate(150 mL) was added. The organic layer was separated, dried (MgSO₄), andthe filtrate was concentrated in vacuo to give a clear oil. The oil wascrystallized from EtOAc/pentane to give the title compound (9.7 g, 86%),FAB-MS 243 (MH⁺).

F) Preparation of N.sup.α t-butyloxycarbonyl-DL-(αMe) ornithine.

A solution of N.sup.α t-butyloxycarbonyl-DL-(α-propionitrile)alanine(10.8 g, 44.4 mmol) in ethyl alcohol (135 mL) was reacted with hydrogenover platinum oxide (3 g) at 60 psi in a Parr shaker apparatus for 24hours at 60° C. The reaction mixture was filtered through a Celite® pad,and the filtrate was concentrated in vacuo. The solid was trituratedwith a mixture of THF, diethyl ether, and pentane. The solid wasfiltered and dried to give pure title compound (8.2 g, 75%).

G) Preparation of N.sup.α t-butyloxycarbonyl-DL-(αMe) arginine.

A solution of N.sup.α t-butyloxycarbonyl-DL-(αMe)ornithine (7.6 g, 30.9mmol) in water (80 mL) was adjusted to pH 10.5 with 2 N NaOH andO-methylisourea hydrogen sulfate (10.6 g, 61.7 mmol) was added. Thereaction was stirred at room temperature for 48 hours. The reaction wascooled to 0° C., and the solid was filtered and dried in vacuo to givepure title compound (5.8 g, 61%), FAB-MS 289 (MH⁺).

H) Preparation of Boc-DL-Arg(αMe) (Cbz)-OH.

A solution of N.sup.α t-butyloxycarbonyl-DL-(αMe)arginine (5.7 g, 18.4mmol) in water (50 mL) was adjusted to pH 13.4 with 5 N NaOH. Thereaction was chilled to -5° C. and the pH was maintained at 13.2-13.5using 5 N NaOH while adding benzyl chloroformate (11 mL, 73.5 mmol)dropwise. The reaction stirred for an additional 1 hour at -5° C. anddiluted with H20 (100 mL) and Et₂ O (100 mL). The aqueous layer wasseparated and extracted with Et₂ O (100 mL). The aqueous layer wasacidified to pH 3.0 with 4 N HCl and extracted with EtOAc (200 mL). Theaqueous layer was extracted twice with EtOAc. The combined EtOAc layerswere washed with water and dried (MgSO₄). The organic solution wasconcentrated to dryness in vacuo to give the title compound (3.9 g,50%), FAB-MS 423 (MH⁺).

Analysis for C₂₀ H₃₀ N₄ O₆ :

Calculated: C, 56.86; H, 7.16; N, 13.26;

Found: C, 56.79; H, 7.18; N, 13.13.

I) Preparation of Boc-DL-Arg(αMe)(Cbz)-lactam.

A solution of Boc-DL-Arg(αMe)(Cbz)-OH (3.76 g, 8.9 mmol) in THF (80 mL)was cooled to -10° C. To the reaction mixture was added triethylamine(1.3 mL, 9.3 mmol) followed by isobutyl chloroformate (1.22 mL, 9.3mmol). The reaction was stirred 5 minutes at -10° C. and an additionalamount of triethylamine (1.3 mL, 9.3 mmol) was added. The reactionstirred for 1 hour at -10° C. and 24 hours at room temperature. Thereaction was poured into 200 mL of ice-water and the resultingprecipitate was filtered, washed with cold water, and the solid dried invacuo. The solid was crystallized from diethyl ether to give pure titlecompound (3.4 g, 95%), FAB-MS 405 (MH⁺).

Analysis for C₂₀ H₂₈ N₄ O₅ :

Calculated: C, 59.39; H, 6.98; N, 13.85;

Found: C, 59.65; H, 7.16; N, 13.16.

J) Preparation of DL-Arg(αMe) (Cbz) -Lactam.HCl.

A solution of EtOAc (20 mL) saturated with gaseous HCl was added to asolution of Boc-DL-Arg(αMe)(Cbz)-lactam (3.03 g, 7.5 mmol) dissolved inCH₂ Cl₂ (10 mL) at room temperature. The reaction was allowed to stir 30minutes at room temperature. The resulting precipitate was filteredwashed with diethyl ether, and dried in vacuo to give the pure titlecompound (2.58 g, 100%), FAB-MS 305 (MH⁺).

K) Preparation of Cbz-D-MePhe-Pro-Arg(αMe)(Cbz)-Lactam.

A solution Cbz-D-MePhe-Pro-OH (1.64 g, 3.2 mmol) in DMF (40 mL) wascooled to 0° C. To the solution was added 1-hydroxybenzotriazole (0.44g, 3.2 mmol), dicyclohexyl -carbodiimide (0.67 g, 3.2 mmol),DL-Arg(αMe)(Cbz)-Lactam.HCl (1.1 g, 3.2 mmol), anddiisopropyl-ethylamine (0.84 mL, 4.8 mmol). The reaction was stirred for1 hour at 0° C. and 48 hours at room temperature. The reactionprecipitate was filtered and the filtrate concentrated in vacuo to anoil. The oil was dissolved in EtOAc (200 mL) and water (100 mL). Theorganic layer was separated, and washed sequentially with 1 N NaHCO₃,water, 1.5 N citric acid, and water. The organic layer was dried(MgSO₄), and the filtrate evaporated to an amorphous solid. The crudesolid was dissolved in chloroform and chromatographed over silica gel. Astep gradient system consisting of (A) chloroform and (B) acetonitrilewas used to elute the correct diastereomeric peptide. The gradient usedwas an increasing concentration of CH₃ CN from 0% to 50%. Fractions werecollected and pooled on the basis of TLC profile. The combined fractionswere concentrated in vacuo to give a clear oil of the title compound(0.71 g, 32%), FAB-MS 697 (MH⁺).

L) Preparation of Cbz-D-MePhe-Pro-Arg(αMe) (Cbz)-H.

Cbz-D-MePhe-Pro-Arg(αMe)(Cbz)-Lactam (0.67 g, 0.962 mmol) was dissolvedin anhydrous THF (35 mL) and placed in a flask under an inertatmosphere. The reaction cooled to -65° C. and lithium aluminum hydride1M in THF (1.0 mL, 1.0 mmol) was added dropwise. The reaction wasstirred for 35 minutes at -65° C. A solution of 5 mL of THF and 0.8 mLof 0.5 N H₂ SO₄ was added dropwise and the reaction was diluted withEtOAc (100 mL) and water (50 mL). The organic layer was separated,washed once with water, and dried (MgSO₄). The filtrate was concentratedto dryness in vacuo to an amorphous solid to give the title compound(0.74 g, 115%): TLC R_(f) (A) 0.45.

M) Preparation of D-MePhe-Pro-Arg((αMe)-H.

Cbz-D-MePhe-Pro-Arg(αMe)(Cbz)-H (0.74 g, 1.05 mmol) was dissolved inethanol (75 mL), water (25 mL), and 1 N HCl (1.9 mL, 1.9 mmol), andhydrogenated in the presence of 5% Pd/C catalyst (0.45 g) at ambienttemperature and pressure. After the reaction was completed, the catalystwas removed by filtration through a Hyflo® pad. The filtrate wasconcentrated in vacuo down to 20 mL and water (50 mL) was added. The pHof solution adjusted to 4.0 with BioRad AG1-X8 resin® (hydroxide form).The resin was removed by filtration and the solution lyophilized to givepure title compound as the dihydrochloride monohydrate (0.436 g, 90%),FAB-MS 431 (MH⁺); [α]_(D) =102.9° (C=0.5/0.01 N HCl).

Analysis for C₂₂ H₃₄ N₆ O₃.2HCl.1H₂ O:

Calculated: C, 50.67; H, 7.34; N, 16.12;

Found: C, 51.07; H, 6.90; N, 15.88.

EXAMPLE 2 D-1-(1,2,3,4-tetrahydroisoquinolinylcarboxyl)-L-prolinyl-L-α-methylarginine aldehyde (D-1-Tiq-Pro-Arg(αMe)-H) ##STR17##

Following the procedure described above for Example 1, the titlecompound was prepared as the dihydrochloride salt. FAB-MS 429 (MH⁺);[α]_(D) =-36.1° (C=0.5/0.01 N HCl).

Analysis for C₂₂ H₃₂ N₆ O₃.2HCl:

Calculated: C, 52.69; H, 6.83; N, 16.76;

Found: C, 52.40; H, 6.61; N, 16.57.

The compounds of the invention are believed to selectively inhibitthrombin over other proteinases and nonenzyme proteins involved in bloodcoagulation without appreciable interference with the body's naturalclot lysing ability (the compounds have a low inhibitory effect onfibrinolysis). Further, such selectivity is believed to permit use withthrombolytic agents without substantial interference with thrombolysisand fibrinolysis. Further, the compounds of the present invention arebelieved to be orally active.

The invention in one of its aspects provides a method of inhibitingthrombin in mammals comprising administering to a mammal in need oftreatment an effective (thrombin inhibiting) dose of a compound offormula I.

The thrombin inhibition contemplated by the present method includes bothmedical therapeutic and/or prophylactic treatment as appropriate.

In a further embodiment the invention relates to treatment, in a humanor animal, of conditions where inhibition of thrombin is required. Thecompounds of the invention are expected to be useful in animals,including man, in treatment or prophylaxis of thrombosis andhypercoagulability in blood and tissues. Disease states in which thecompounds have a potential utility are in treatment or prophylaxis ofthrombosis and hypercoagulability in blood and tissues. Disease statesin which the compounds have a potential utility, in treatment and/orprophylaxis, include venous thrombosis and pulmonary embolism, arterialthrombosis, such as in myocardial ischemia, myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis. Further, the compounds have expected utility in prophylaxisof atherosclerotic diseases such as coronary arterial disease, cerebralarterial disease and peripheral arterial disease. Further, the compoundsare expected to be useful together with thrombolytics in myocardialinfarction. Further, the compounds have expected utility in thetreatment or prophylaxis for reocclusion after thrombolysis,percutaneous transluminal angioplasty (PTCA) and coronary bypassoperations. Further, the compounds have expected utility in preventionof rethrombosis after microsurgery. Further, the compounds are expectedto be useful in anticoagulant treatment in connection with artificialorgans and cardiac valves. Further, the compounds have expected utilityin anticoagulant treatment in hemodialysis and disseminatedintravascular coagulation. A further expected utility is in rinsing ofcatheters and mechanical devices used in patients in vivo, and as ananticoagulant for preservation of blood, plasma and other blood productsin vitro. Still further, the compounds have expected utility in otherdiseases where blood coagulation could be a fundamental contributingprocess or a source of secondary pathology, such as cancer, includingmetastasis, and inflammatory diseases, including arthritis and diabetes.The anti-coagulant compound is administered orally, or parenterally,e.g., by intravenous infusion (iv), intramuscular injection (im) orsubcutaneously (sc).

The specific dose of a compound administered according to this inventionto obtain therapeutic and/or prophylactic effects will, of course, bedetermined by the particular circumstances surrounding the case,including, for example, the compound administered, the rate ofadministration, and the condition being treated.

A typical daily dose for each of the above utilities is between about0.01 mg/kg and about 1000 mg/kg. The dose regime may vary e.g. forprophylactic use a single daily dose may be administered or multipledoses such as 3 or 5 times daily may be appropriate. In critical caresituations a compound of the invention is administered by iv infusion ata rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferablybetween about 0.1 mg/kg/h and about 5 mg/kg/h.

The method of this invention also is practiced in conjunction with aclot lysing agent, e.g., tissue plasminogen activator (tPA), modifiedtPA, streptokinase or urokinase. In cases when clot formation hasoccurred and an artery or vein is blocked, either partially or totally,a clot lysing agent is usually employed. A compound of the invention canbe administered prior to or along with the lysing agent or subsequent toits use alone and preferably further is administered along with aspirinto prevent the reoccurrence of clot formation.

The method of this invention is also practiced in conjunction with aplatelet glycoprotein receptor (IIb/IIIa) antagonist, that inhibitsplatelet aggregation. A compound of the invention can be administeredprior to or along with the IIb/IIIa antagonist or subsequent to its useto prevent the reoccurrence of clot formation.

The method of this invention is also practiced in conjunction withaspirin. A compound of the invention can be administered prior to oralong with aspirin or subsequent to its use to prevent the reoccurrenceof clot formation. As stated above, preferably a compound of the presentinvention is administered in conjunction with a clot lysing agent andaspirin.

This invention also provides pharmaceutical formulations for use in theabove described therapeutic method. Pharmaceutical formulations of theinvention comprise an effective thrombin inhibiting amount of a compoundof Formula I in association with a pharmaceutically acceptable carrier,excipient or diluent. For oral administration the antithromboticcompound is formulated in gelatin capsules or tablets which may containexcipients such as binders, lubricants, disintegration agents and thelike. For parenteral administration the antithrombotic is formulated ina pharmaceutically acceptable diluent, e.g., physiological saline(0.9%), 5% dextrose, Ringer's solution, and the like.

The compound of the present invention can be formulated in unit dosageformulations comprising a dose between about 0.1 mg and about 1000 mg.Preferably the compound is in the form of a pharmaceutically acceptablesalt such as for example the sulfate salt, acetate salt or a phosphatesalt. An example of a unit dosage formulation comprises 5 mg of acompound of the present invention as a pharmaceutically acceptable saltin a 10 mL sterile glass ampoule. Another example of a unit dosageformulation comprises about 10 mg of a compound of the present inventionas a pharmaceutically acceptable salt in 20 mL of isotonic salinecontained in a sterile ampoule.

The compounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. The compounds of the present invention are preferablyformulated prior to administration. Therefore, another embodiment of thepresent invention is a pharmaceutical formulation comprising aneffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt or solvate thereof in association with apharmaceutically acceptable carrier, diluent or excipient therefor.

The active ingredient in such formulations comprises from 0.1% to 99.9%by weight of the formulation. By "pharmaceutically acceptable" it ismeant the carrier, diluent or excipient must be compatible with theother ingredients of the formulation and not deleterious to therecipient thereof. The present pharmaceutical formulations are preparedby known procedures using well known and readily available ingredients.In making the compositions of the present invention, the activeingredient will usually be admixed with a carrier, or diluted by acarrier, or enclosed within a carrier which may be in the form of acapsule, sachet, paper or other container. When the carrier serves as adiluent, it may be a solid, semi-solid or liquid material which acts asa vehicle, excipient or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols, (as a solid or in a liquid medium), soft and hard gelatincapsules, suppositories, sterile injectable solutions, sterile packagedpowders, and the like. The compositions of this invention may beformulated so as to provide quick, sustained, or delayed release of theactive ingredient after administration to the patient by employingprocedures well known in the art.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way. "Activeingredient," of course, means a compound according to Formula I or apharmaceutically acceptable salt or solvate thereof.

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

    ______________________________________                                                             Quantity                                                                      (mg/                                                                          capsule)                                                 ______________________________________                                        Active ingredient      250                                                    Starch, dried          200                                                    Magnesium stearate     10                                                     Total                  460    mg                                              ______________________________________                                    

Formulation 2

A tablet is prepared using the ingredients below:

    ______________________________________                                                              Quantity                                                                      (mg/                                                                          capsule)                                                ______________________________________                                        Active ingredient       250                                                   Cellulose, microcrystalline                                                                           400                                                   Silicon dioxide, fumed  10                                                    Stearic acid            5                                                     Total                   665    mg                                             ______________________________________                                    

The components are blended and compressed to form tablets each weighing665 mg

Formulation 3

An aerosol solution is prepared containing the following components:

    ______________________________________                                                             Weight                                                   ______________________________________                                        Active ingredient      0.25                                                   Ethanol                25.75                                                  Propellant 22 (Chlorodifluoromethane)                                                                70.00                                                  Total                  100.00                                                 ______________________________________                                    

The active compound is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to -30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4

Tablets, each containing 60 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient         60      mg                                          Starch                    45      mg                                          Microcrystalline cellulose                                                                              35      mg                                          Polyvinylpyrrolidone (as 10% solution in water)                                                         4       mg                                          Sodium carboxymethyl starch                                                                             4.5     mg                                          Magnesium stearate        0.5     mg                                          Talc                      1       mg                                          Total                     150     mg                                          ______________________________________                                    

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The aqueous solution containingpolyvinyl-pyrrolidone is mixed with the resultant powder, and themixture then is passed through a No. 14 mesh U.S. sieve. The granules soproduced are dried at 50° C. and passed through a No. 18 mesh U.S.Sieve. The sodium carboxymethyl starch, magnesium stearate and talc,previously passed through a No. 60 mesh U.S. sieve, are then added tothe granules which, after mixing, are compressed on a tablet machine toyield tablets each weighing 150 mg.

Formulation 5

Capsules, each containing 80 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient       80     mg                                             Starch                  59     mg                                             Microcrystalline cellulose                                                                            59     mg                                             Magnesium stearate      2      mg                                             Total                   200    mg                                             ______________________________________                                    

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve, and filled into hardgelatin capsules in 200 mg quantities.

Formulation 6

Suppositories, each containing 225 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient       225    mg                                             Saturated fatty acid glycerides                                                                       2,000  mg                                             Total                   2,225  mg                                             ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7

Suspensions, each containing 50 mg of active ingredient per 5 mL dose,are made as follows:

    ______________________________________                                        Active ingredient       50     mg                                             Sodium carboxymethyl cellulose                                                                        50     mg                                             Syrup                   1.25   mL                                             Benzoic acid solution   0.10   mL                                             Flavor                  q.v.                                                  Color                   q.v.                                                  Purified water to total 5      mL                                             ______________________________________                                    

The active ingredient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form a smoothpaste. The benzoic acid solution, flavor and color are diluted with aportion of the water and added, with stirring. Sufficient water is thenadded to produce the required volume.

Formulation 8.

An intravenous formulation may be prepared as follows:

    ______________________________________                                        Active ingredient     100    mg                                               Isotonic saline       1,000  mL                                               ______________________________________                                    

The solution of the above ingredients generally is administeredintravenously to a subject at a rate of 1 mL per minute.

The compounds provided by the invention (Formula I) are orally activeand selectively inhibit the action of thrombin in mammals.

The ability of the compounds of the present invention to be an effectiveand orally active thrombin inhibitor is evaluated in one or more of thefollowing assays.

The inhibition of thrombin is demonstrated by in vitro inhibition of theamidase activity of thrombin as measured in an assay in which thrombinhydrolyzes the chromogenic substrate,N-benzoyl-L-phenylalanyl-L-valyl-L-arginyl-p-nitroanilide.

The assay is carried out by mixing 50 μL buffer (0.03M Tris, 0.15M NaCl,pH 7.4) with 25 μL of bovine thrombin or human thrombin solution (0.21mg/mL of thrombostat bovine thrombin, Parke-Davis, or purified humanthrombin, Enzyme Research Laboratories, South Bend, Ind., at about 8 NIHunits/mL, in the same buffer) and 25 μL of test compound in a solvent(in 50% aqueous methanol, v:v). The 150 μL of an aqueous solution of thechromogenic substrate (at 0.25 mg/mL) are added and the rates ofhydrolysis of the substrate are measured by monitoring the reactions at405 nm for the release of p-nitroaniline. Standard curves areconstructed by plotting free thrombin concentration against hydrolysisrate. The hydrolysis rates observed with test compounds are thenconverted to "free thrombin" values in the respective assays by use ofthe standard curves. The bound thrombin (bound to test compound) iscalculated by subtracting the amount of free thrombin observed in eachassay from the known initial amount of thrombin used in the assay. Theamount of free inhibitor in each assay is calculated by subtracting thenumber of moles of bound thrombin from the number of moles of addedinhibitor (test compound).

The Kass value is the hypothetical equilibrium constant for the reactionbetween thrombin and the test compound (I). ##EQU1##

Kass is calculated for a range of concentrations of test compounds andthe mean value is reported in units of liter per mole.

By substantially following the procedures described above for humanthrombin, and using other human blood coagulation system serineproteases and proteases of the fibrinolytic system with the appropriatechromogenic substrates, identified below, selectivity of the compoundsof the present invention with respect to the coagulation factor serineproteases and with respect to the fibrinolytic system serine proteasesare evaluated as well as their substantial lack of interference withserine proteases of the fibrinolytic system. Thrombin inhibitorspreferably should spare fibrinolysis induced by urokinase, tissueplasminogen activator (t-PA) and streptokinase. This would be importantto the therapeutic use of such agents as an adjunct to streptokinase,t-PA or urokinase thrombolytic therapy and to the use of such agents asan endogenous fibrinolysis-sparing (with respect to t-PA and urokinase)antithrombotic agent. In addition to the lack of interference with theamidase activity of the fibrinolytic proteases, such fibrinolytic systemsparing can be studied by the use of human plasma clots and their lysisby the respective fibrinolytic plasminogen activators.

Human factors X, Xa, IXa, XIa, and XIIa are purchased from EnzymeResearch Laboratories, South Bend, Ind.; human urokinase from LeoPharmaceuticals, Denmark; and recombinant activated Protein C (aPC) isprepared at Eli Lilly and Co. substantially according to U.S. Pat. No.4,981,952 incorporated by reference herein in its entirety. Chromogenicsubstrates: N-Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (for factor Xa);N-Cbz-D-Arg-Gly-Arg-p-nitroanilide (for factor IXa assay as the factorXa substrate); pyroglutamyl-Pro-Arg-p-nitroanilide (for Factor XIa andfor aPC); H-D-Pro-Phe-Arg-p-nitroanilide (for factor XIIa); andpyroglutamyl-Gly-Arg-p-nitroanilide (for urokinase); are purchased fromKabiVitrum, Stockholm, Sweden, or from Midwest Biotech, Fishers, Ind..Bovine trypsin is purchased from Worthington Biochemicals, Freehold,N.J., and human plasma kallikrein from Kabi Vitrum, Stockholm, Sweden.Chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide for plasmakallikrein is purchased from Kabi Vitrum, Stockholm, Sweden.N-Benzoyl-Phe-Val-Arg-p-nitroanilide, the substrate for human thrombinand for trypsin, is synthesized according to procedures described abovefor the compounds of the present invention, using known methods ofpeptide coupling from commercially available reactants or purchased fromMidwest Biotech, Fishers, Ind..

Human plasmin is purchased from Boehringer Mannheim, Indianapolis, Ind.;nt-PA is purchased as single chain activity reference from AmericanDiagnostica, Greenwich, Conn.; modified-t-PA6 (mt-PA6) is prepared atEli Lilly and Company by procedure known in the art (See, Burck, et al.,J. Biol. Chem., 265, 5120-5177 (1990). Plasmin chromogenic substrateH-D-Val-Leu-Lys-p-nitroanilide and tissue plasminogen activator (t-PA)substrate H-D-Ile-Pro-Arg-p-nitroanilide are purchased from Kabi Vitrum,Stockholm, Sweden.

In the chromogenic substrates described above the three-letter symbolsIle, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys are used to indicate thecorresponding amino acid group isoleucine, glutamic acid, glycine,proline, arginine, phenylalanine, valine, leucine and lysine,respectively.

Table 1 which follows lists the Kass values obtained with the indicatedcompound represented by the Formula I.

                  TABLE 1                                                         ______________________________________                                        Inhibition Properties                                                         Kass × 10.sup.6 (L/mole)                                                        Bovine                                                                Example Thrombin  Trypsin   Plasmin                                                                              Xa     t-PA                                ______________________________________                                        1       1.1       0.004     0.0005 0.0003 0                                   2       0.22      0.014     0.0008 0.001  0                                   ______________________________________                                    

Materials

Dog plasma is obtained from conscious mixed-breed hounds (either sexHazelton-LRE, Kalamazoo, Mich., U.S.A.) by venipuncture into 3.8 percentcitrate. Fibrinogen is prepared from fresh dog plasma and humanfibrinogen is prepared from in-date ACD human blood at the fraction I-2according to previous procedures and specifications. Smith, Biochem. J.,185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967,(1972). Human fibrinogen (98 percent pure/plasmin free) is from AmericanDiagnostica, Greenwich, Conn. Radiolabeling of fibrinogen I-2preparations is performed as previously reported. Smith, et al.,Biochemistry, 11, 2958-2967, (1972). Urokinase is purchased form LeoPharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase ispurchased from Hoechst-Roussel Pharmaceuticals, Somerville, N.J.

Methods-Effects on Lysis of Human Plasma Clots by t-PA

Human plasma clots are formed in micro test tubes by adding 50 μLthrombin (73 NIH unit/mL) to 100 μL human plasma which contains 0.0229μCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlayingthe clots with 50 μL of urokinase or streptokinase (50, 100, or 1000unit/mL) and incubating for 20 hours at room temperature. Afterincubation the tubes are centrifuged in a Beckman Microfuge. 25 μL ofsupernate is added into 1.0 mL volume of 0.03M tris/0.15M NaCl bufferfor gamma counting. Counting controls 100 percent lysis are obtained byomitting thrombin (and substituting buffer). The thrombin inhibitors areevaluated for possible interference with fibrinolysis by including thecompounds in the overlay solutions at 1, 5, and 10 ug/mL concentrations.Rough approximations of IC50 values are estimated by linearextrapolations from data points to a value which would represent 50percent of lysis for that particular concentration of fibrinolyticagent.

Anticoagulant Activity Materials

Dog plasma and rat plasma is obtained from conscious mixed-breed hounds(either sex, Hazelton-LRE, Kalamazoo, Mich., U.S.A.) or fromanesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc.,Indianapolis, Ind., U.S.A.) by venipuncture into 3.8 percent citrate.Fibrinogen is prepared from in-date ACD human blood as the fraction I-2according to previous procedures and specifications. Smith, Biochem. J.,185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967 (1972).Human fibrinogen is also purchased as 98 percent pure/plasmin free fromAmerican Diagnostica, Greenwich, Conn. Coagulation reagents ACTIN,Thromboplastin, and Human plasma are from Baxter Healthcare Corp., DadeDivision, Miami, Fla. Bovine thrombin from Parke-Davis (Ann Detroit,Mich.) is used for coagulation assays in plasma.

Methods Anticoagulation Determinations

Coagulation assay procedures are as previously described. Smith, et al.,Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulationinstrument (American LABor, Inc.) is used for all coagulation assaymeasurements. The prothrombin time (PT) is measured by adding 0.05 mLsaline and 0.05 mL Thromboplastin-C reagent to 0.05 mL test plasma. Theactivated partial thromboplastin time (APTT) is measured by incubationof 0.05 mL test plasma with 0.05 mL Actin reagent for 120 secondsfollowed by 0.05 mL CaCl₂ (0.02M). The thrombin time (TT) is measured byadding 0.05 mL saline and 0.05 mL thrombin (10 NIH units/mL) to 0.05 mLtest plasma. The compounds of formula I are added to human or animalplasma over a wide range of concentrations to determine prolongationeffects on the APTT, PT, and TT assays. Linear extrapolations areperformed to estimate the concentrations required to double the clottingtime for each assay.

Animals

Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc.,Indianapolis, Ind.) are anesthetized with xylazine (20 mg/kg, s.c.) andketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37°C.). The jugular vein(s) is cannulated to allow for infusions.

Arterio-Venous shunt model

The left jugular vein and right carotid artery are cannulated with 20 cmlengths of polyethylene PE 60 tubing. A 6 cm center section of largertubing (PE 190) with a cotton thread (5 cm) in the lumen, is frictionfitted between the longer sections to complete the arterio-venous shuntcircuit. Blood circulated through the shunt for 15 minutes before thethread is carefully removed and weighed. The weight of a wet thread issubtracted from the total weight of the thread and thrombus (see J. R.Smith, Br. J. Pharmacol., 77,29 (1982)).

FeCl₃ model of arterial injury

The carotid arteries are isolated via a midline ventral cervicalincision. A thermocouple is placed under each artery and vesseltemperature is recorded continuously on a strip chart recorder. A cuffof tubing (0.058 ID×0.077 OD×4 mm, Baxter Med. Grade Silicone), cutlongitudinally, is placed around each carotid directly above thethermocouple. FeCl₃ hexahydrate is dissolved in water and theconcentration (20%) is expressed in terms of the actual weight of FeCl₃only. To injure the artery and induce thrombosis, 2.85 μl is pipettedinto the cuff to bathe the artery above the thermocouple probe. Arterialocclusion is indicated by a rapid drop in temperature. The time toocclusion is reported in minutes and represented the elapsed timebetween application of FeCl₃ and the rapid drop in vessel temperature(see K. D. Kurz, Thromb. Res., 60, 269 (1990)).

Spontaneous thrombolysis model

In vitro data suggested that the peptide thrombin inhibitors inhibitthrombin and other serine proteases, such as plasmin and tissueplasminogen activator. To assess if the compounds inhibited fibrinolysisin vivo, the rate of spontaneous thrombolysis is determined byimplanting a labeled whole blood clot into the pulmonary circulation.Rat blood (1 mL) is mixed rapidly with bovine thrombin (4 IU, ParkeDavis) and ¹²⁵ I human fibrogen (5 μCi, ICN), immediately drawn intosilastic tubing and incubated at 37° C. for 1 hour. The aged thrombus isexpelled from the tubing, cut into 1 cm segments, washed 3× in normalsaline and each segment is counted in a gamma counter. A segment withknown counts is aspirated into a catheter that is subsequently implantedinto the jugular vein. The catheter tip is advanced to the vicinity ofthe right atrium and the clot is expelled to float into the pulmonarycirculation. One hour after implant, the heart and lungs are harvestedand counted separately. Thrombolysis is expressed as a percentage where:##EQU2## The fibrinolytic dissolution of the implanted clot occurstime-dependently (see J. P. Clozel, Cardiovas. Pharmacol., 12, 520(1988)).

Coagulation parameters

Plasma thrombin time (TT) and activated partial thromboplastin time(APTT) are measured with a fibrometer. Blood is sampled from a jugularcatheter and collected in syringe containing sodium citrate (3.8%, 1part to 9 parts blood). To measure TT, rat plasma (0.1 mL) is mixed withsaline (0.1 mL) and bovine thrombin (0.1 mL, 30 U/mL in TRIS buffer;Parke Davis) at 37° C. For APTT, plasma (0.1 mL) and APTT solution (0.1mL, Organon Teknika) are incubated for 5 minutes (37° C.) and CaCl₂(0.01 mL, 0.025 M) is added to start coagulation. Assays are done induplicate and averaged.

Index of Bioavailability

A measure of bioactivity, plasma thrombin time (TT), served as asubstitute for the assay of parent compound on the assumption thatincrements in TT resulted from thrombin inhibition by parent only. Thetime course of the effect of the thrombin inhibitor upon TT isdetermined after i.v. bolus administration to anesthetized rats andafter oral treatment of fasted conscious rats. Due to limitations ofblood volume and the number of points required to determine the timecourse from time of treatment to the time when the response returned topretreatment values, two populations of rats are used. Each samplepopulation represented alternating sequential time points. The averageTT over the time course is used to calculate area under the curve (AUC).The index of bioavailability is calculated by the formula shown belowand is expressed as percent relative activity.

The area under the curve (AUC) of the plasma TT time course isdetermined and adjusted for the dose. This index of bioavailability istermed "% Relative Activity" and is calculated as ##EQU3##

Compounds

Compound solutions are prepared fresh daily in normal saline and areinjected as a bolus or are infused starting 15 minutes before andcontinuing throughout the experimental perturbation which is 15 minutesin the arteriovenous shunt model and 60 minutes in the FeCl₃ model ofarterial injury and in the spontaneous thombosis model. Bolus injectionvolume is 1 mL/kg for i.v., and 5 mL/kg for p.o. and infusion volume is3 mL/hr.

Statistics

Results are expressed as means +/- SEM. One-way analysis of variance isused to detect statistically significant differences and then Dunnett'stest is applied to determine which means are different. Significancelevel for rejection of the null hypothesis of equal means is P<0.05.

Animals

Male dogs (Beagles; 18 months-2 years; 12-13 kg, Marshall Farms, NorthRose, N.Y. 14516) are fasted overnight and fed Purina certifiedPrescription Diet (Purina Mills, St. Louis, Mo.) 240 minutes afterdosing. Water is available ad libitum. The room temperature ismaintained between 66°-74° F.; 45-50% relative humidity; and lightedfrom 0600-1800 hours.

Pharmacokinetic model

Test compound is formulated immediately prior to dosing by dissolving insterile 0.9% saline to a 5 mg/mL preparation. Dogs are given a single 2mg/kg dose of test compound by oral gavage. Blood samples (4.5 mL) aretaken from the cephalic vein at 0.25, 0.5, 0.75, 1, 2, 3, 4 and 6 hoursafter dosing. Samples are collected in citrated Vacutainer tubes andkept on ice prior to reduction to plasma by centrifugation. Plasmasamples are derivatized with dinitrophenylhydrazine and analyzed by HPLC(Zorbax SB-C8 column) eluting with methanol/500 mM sodium acetateadjusted to pH 7 with phosphoric acid (60:40, v/v). Plasma concentrationof test compound is recorded and used to calculate the pharmacokineticparameters: elimination rate constant, Ke; total clearance, Clt; volumeof distribution, V_(D) ; time of maximum plasma test compoundconcentration, Tmax; maximum concentration of test compound at Tmax,Cmax; plasma half-life, to 0.5; area under the curve, A.U.C.; andfraction of test compound absorbed, F.

Canine Model of Coronary Artery Thrombosis

Surgical preparation and instrumentation of the dogs are as described inJackson, et al., Circulation, 82, 930-940 (1990). Mixed-breed hounds(aged 6-7 months, either sex, Hazelton-LRE, Kalamazoo, Mich., U.S.A.)are anesthetized with sodium pentobarbital (30 mg/kg intravenously,i.v.), intubated, and ventilated with room air. Tidal volume andrespiratory rates are adjusted to maintain blood PO₂, PCO₂, and pHwithin normal limits. Subdermal needle electrodes are inserted for therecording of a lead II ECG.

The left jugular vein and common carotid artery are isolated through aleft mediolateral neck incision. Arterial blood pressure (ABP) ismeasured continuously with a precalibrated Millar transducer (modelMPC-500, Millar Instruments, Houston, Tex., U.S.A.) inserted into thecarotid artery. The jugular vein is cannulated for blood sampling duringthe experiment. In addition, the femoral veins of both hindlegs arecannulated for administration of test compound.

A left thoracotomy is performed at the fifth intercostal space, and theheart is suspended in a pericardial cradle. A 1- to 2-cm segment of theleft circumflex coronary artery (LCX) is isolated proximal to the firstmajor diagonal ventricular branch. A 26-gauge needle-tipped wire anodalelectrode (Teflon®-coated, 30-gauge silverplated copper wire) 3-4 mmlong is inserted into the LCX and placed in contact with the intimalsurface of the artery (confirmed at the end of the experiment). Thestimulating circuit is completed by placing the cathode in asubcutaneous (s.c.) site. An adjustable plastic occluder is placedaround the LCX, over the region of the electrode. A precalibratedelectromagnetic flow probe (Carolina Medical Electronics, King, N.C.,U.S.A.) is placed around the LCX proximal to the anode for measurementof coronary blood flow (CBF). The occluder is adjusted to produce a40-50% inhibition of the hyperemic blood flow response observed after10-s mechanical occlusion of the LCX. All hemodynamic and ECGmeasurements are recorded and analyzed with a data acquisition system(model M3000, Modular Instruments, Malvern, Pa. U.S.A.).

Thrombus Formulation and Compound Administration Regimens

Electrolytic injury of the intima of the LCX is produced by applying100-μA direct current (DC) to the anode. The current is maintained for60 minutes and then discontinued whether the vessel has occluded or not.Thrombus formation proceeds spontaneously until the LCX is totallyoccluded (determined as zero CBF and an increase in the S-T segment).Compound administration is started after the occluding thrombus isallowed to age for 1 hour. A 2-hour infusion of the compounds of thepresent invention at doses of 0.5 and 1 mg/kg/h is begun simultaneouslywith an infusion of thrombotic agent (e.g. tissue plasminogen activator,streptokinase, APSAC). Reperfusion is followed for 3 hours afteradministration of test compound. Reocclusion of coronary arteries aftersuccessful thrombolysis is defined as zero CBF which persisted for ≧30minutes.

Hematology and template bleeding time determinations

Whole blood cell counts, hemoglobin, and hematocrit values aredetermined on a 40-μl sample of citrated (3.8%) blood (1 part citrate:9parts blood) with a hematology analyzer (Cell-Dyn 900, Sequoia-Turner.Mount View, Calif., U.S.A.). Gingival template bleeding times aredetermined with a Simplate II bleeding time device (Organon TeknikaDurham, N.C., U.S.A.). The device is used to make 2 horizontal incisionsin the gingiva of either the upper or lower left jaw of the dog. Eachincision is 3 mm wide×2 mm deep. The incisions are made, and a stopwatchis used to determine how long bleeding occurs. A cotton swab is used tosoak up the blood as it oozes from the incision. Template bleeding timeis the time from incision to stoppage of bleeding. Bleeding times aretaken just before administration of test compound (0 minutes), 60minutes into infusion, at conclusion of administration of the testcompound (120 minutes), and at the end of the experiment.

All data are analyzed by one-way analysis of variance (ANOVA) followedby Student-Neuman-Kuels post hoc t test to determine the level ofsignificance. Repeated-measures ANOVA are used to determine significantdifferences between time points during the experiments. Values aredetermined to be statistically different at least at the level ofp<0.05. All values are mean±SEM. All studies are conducted in accordancewith the guiding principles of the American Physiological Society.Further details regarding the procedures are described in Jackson, etal., J, Cardiovasc. Pharmacol., 21, 587-599 (1993).

We claim:
 1. A compound having the formula ##STR18## wherein X is##STR19## Q is --OH, C₁ -C₄ alkoxy, or --NH--A; A is hydrogen, C₁ -C₄alkyl, acetyl, CF₃ C(O)--, CF₃ CF₂ C(O)--, R"SO₂ --, benzyloxycarbonyl,or t-butyloxycarbonyl;R' is hydrogen, C₁ -C₄ alkyl, phenyl, or benzyl;R" is C₁ -C₄ alkyl, --(CH₂)_(d) --COOH, or unsubstituted or substitutedaryl or heteroaryl, where aryl is phenyl or naphthyl, and heteroaryl isa 5- or 6-membered unsubstituted or substituted aromatic heterocyclicring, having one or two heteroatoms which are the same or different andwhich are selected from sulfur, oxygen and nitrogen, or a 9- or10-membered unsubstituted or substituted fused bicyclic aromaticheterocyclic group having one or two heteroatoms which are the same ordifferent and which are selected from sulfur, oxygen and nitrogen; d is1, 2, or 3; m is 0, 1, or 2; n is 0, 1, or 2; Y is ##STR20## R is methylor ethyl; and Z is hydrogen, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, hydroxy, halo,or R_(a) SO₂ NH--, where R_(a) is C₁ -C₄ alkyl; or a pharmaceuticallyacceptable salt thereof or a pharmaceutically acceptable solvate of saidcompound of formula I or salt thereof.
 2. A compound of claim 1 where Xis MePhe, 1- or 3-Tiq, or 1- or 3-Piq, or a pharmaceutically acceptablesalt thereof or a pharmaceutically acceptable solvate of said compoundor salt thereof.
 3. A compound of claim 2 where R is methyl, or apharmaceutically acceptable salt thereof or a pharmaceuticallyacceptable solvate of said compound or salt thereof.
 4. A compound ofclaim 3 which isD-1-(1,2,3,4-tetrahydroisoquinolinylcarboxyl)-L-prolinyl-L-α-methylargininealdehyde or a pharmaceutically acceptable salt thereof or a solvate ofsaid compound or salt thereof.
 5. A compound of claim 3 which isD-N-methylphenylalanyl-L-prolinyl-L-α-methylarginine aldehyde or apharmaceutically acceptable salt thereof or a solvate thereof.
 6. Apharmaceutical formulation comprising a compound of claim 1 or apharmaceutically acceptable salt thereof or a pharmaceuticallyacceptable solvate of said compound or salt thereof, in association witha pharmaceutically acceptable carrier, diluent, or excipient therefore.7. A formulation of claim 6 where X is MePhe, 1- or 3-Tiq, or 1- or3-Piq.
 8. A formulation of claim 7 where R is methyl.
 9. A formulationof claim 8 where said compound isD-N-methylphenylalanyl-L-prolinyl-L-α-methylarginine aldehyde.
 10. Aformulation of claim 8 where said compound is(1,2,3,4-tetrahydroisoquinolinylcarboxyl) -L-prolinyl-L-α-methylargininealdehyde.
 11. A method of inhibiting thrombin in mammals, comprisingadministering to a mammal requiring thrombin inhibition, an effectivedose of a compound of claim 1 or a pharmaceutically acceptable saltthereof or a pharmaceutically acceptable solvate of said compound orsalt thereof.
 12. The method of claim 11 where X is MePhe, 1- or 3-Tiq,or 1- or 3-Piq.
 13. The method of claim 12 where R is methyl.
 14. Themethod of claim 13 where said compound isD-N-methylphenylalanyl-L-prolinyl-L-α-methylarginine aldehyde.
 15. Themethod of claim 13 where said compound is(1,2,3,4-tetrahydroisoquinolinylcarboxyl)-L-prolinyl-L-α-methylargininealdehyde.